转化生长因子β_1对不同活化状态肝星状细胞增殖与Ⅰ型胶原表达的影响

Effects of transforming growth factor β1 on the proliferation and type I collagen expression at different differential rat hepatic stellate cells

目的观察不同活化状态肝星状细胞 (HSC) 对外源性转化生长因子-β1(TGF-β1)旁分泌刺激的生物学效应作用. 方法原代分离培养大鼠HSC,无包被塑料培养皿上分别培养1、4、7d,细胞处于静止、中间活化与完全活化状态,继以10～500pmol/L TGF-β1温育细胞24h,3H-TdR掺入法测定细胞增殖,western blot法检测细胞α-平滑肌肌动蛋白(α-SMA)与I型胶原蛋白表达沉积,3H-脯氨酸掺入与胶原酶消化法测定细胞总胶原的分泌量.100pmol/L TGF-β1温育细胞15～90min,northern blot法检测细胞I型前胶原mRNA的表达水平. 结果 TGF-β1浓度依赖性抑制培养1d HSC的细胞增殖,10～500pmol/L TGF-β1浓度组细胞内3H-TdR掺入率分别为对照组的52.8%～16.8%,与对照组比较,q值为5.44～10.37,P<0.01.但TGF-β1对培养4d与7d的细胞增殖无影响.随细胞活化,HSC基础性α-SMA、I型胶原蛋白与mRNA水平明显增加,而TGF-β1刺激各培养时间HSC以上蛋白与基因的表达.培养1、4、7d HSC基础水平与TGF-β1刺激的总胶原分泌量分别为(804±274)dpm/孔与(1200±708)dpm/孔;(2966±1701)dpm/孔与(6160±1123)dpm/孔;(2580±767)dpm/孔与(4583±1467)dpm/孔,后2组组内比较,t值分别为3.84与2.96,P<0.01或P<0.05.以培养4d HSC的总胶原分泌与α-SMA表达的效应增加较为明显. 结论 TGF-β1明显抑制静止HSC的细胞增殖,对活化状态HSC生长无影响,促进各种活化状态HSC的表型活化与胶原生成,TGF-β1对活化过程中HSC的增殖与胶原生成功能发生岐化.中间活化状态HSC对TGF-β1促表型活化与胶原生成最为明显,可以作为较理想的药理研究细胞模型.

Objective To investigate the biological responses of cultured hepatic stellate cells (HSC) at different differentiated stages on exotic transforming growth factor (TGF-β1). Methods HSC was isolated from rat and primarily cultured in uncoated disc for 1 d, 4 d and 7 d, when the cells were at quiescent, intermediate activated and full activated stages respectively. The cells were incubated with 10 pmol/L- 500 pmol/L TGF-β1 for 24 h, cell proliferation was measured with [3H] TdR incorporation, a -smooth muscle actin (α-SMA) and type I collagen protein were assayed with Western blot, and total collagen secretion in the culture supernatant was analyzed by [3H] proline pulse and collagenase digestion. HSC was treated with 100 pmol/L TGF- β1 for 15 min-90 min, and type I pro-collagen mRNA level was assayed by Northern blot. Results TGF- β1 remarkably inhibited d1 HSC proliferation, the percentage of [3H] TdR incorporation at 10 pmol/L- 500 pmol/L TGF-β1, was 52.8% - 16.8% of the control, q value was 5.44 - 10.37 and P<0.01 vs control. But TGF-β1 had no influence on d4 and d7 HSC. As the cells cultivation prolonged and activated, the basal levels of α-SMA, type I collagen and gene expression increased gradually. TGF-β1 increased the above protein and gene expression. The basal and TGF- β1 stimulated total collagen secretion levels at dl-d7 HSC were (804± 274) vs (1 200 ?708) dpm/well; (2 966 ± 1 701) vs (6 160 ± 1 123) dpm/well, t = 3.84, P < 0.01; (2 580± 767) vs (4 583± 1 467) dpm/well, t = 2.96, P < 0.05. While d4 HSC showed the strongest response of total protein secretion and α-SMA expression. Conclusions TGF- β1 remarkably inhibited quiescent HSC proliferation, and promoted HSC collagen production at both quiescent and activated HSC. Intermediate HSC had the strongest response to TGF- β1, while activated HSC lost the response to TGF-β1 inhibitory growth, and TGF- β1 exerted divergent actions on HSC as the cells activated.